6-(2&#39;-phenylpyrazine-3&#39;-carboxamido) penicillanic acids and salts thereof



United States Tatent Ofitice 3 311 610 642' rrramrunairwn 3'cAanoxAMrno) PENlCiLLANlC ACilDS AND SALTS THEREOF Talrayuki Naito andSusanna Nalragawa, Tokyo, Japan,

assignors to Bristol-Bangui Research institute, Ltd,

Tokyo, Japan, a corporation of Japan No Drawing. Filed June 8, 1965,Ser. No. 462,389

14 Claims. (Cl. 260-2395.)

This application is a continuation-in-part of our prior, copendingapplication Ser. No. 277,112 filed May 1, 1963, now abandoned.

This invention relates to new synthetic compounds of Value asantibacterial agents, as nutritional supplements in animal feeds, asagents for the treatment of mastitis in cattle and as therapeutic agentsin poultry and animals, including man, in the treatment of infectiousdiseases caused by gram-positive bacteria and, more particularly relatesto novel 6 (2-phenylpyrazine 3' carboxamido)penicillanic acid which maycontain certain substituents in the benzene ring, and nontoxic saltsthereof.

Antibacterial agents of the penicillin class have proven highlyeffective in the therapy of infections due to grampositive bacteria butnearly all such penicillins are ineffective against numerous so-calledresistant strains of bacteria, e.g., benzylpenicillin-resistant strainsof Staphylococcus aureus (Micrococcus pyogenes var. aureus). It is theobject of the present invention to provide novel compounds which areeffective against such resistant strains and are orally active and aredecomposed only slowly by aqueous acid. It is a further object of thepresent invention to provide agents which actually inhibit penicillinaseand are thus also useful adjuvants for penicillins such asbenzylpeniciliin.

The objects of the present invention have been achieved by theprovision, according to the present invention, of a member selected fromthe group consisting of an acid of the formula wherein R and R are eacha member selected from the group consisting of hydrogen,trifiuoromethyl, chloro, bromo, (lower)aikylthio, (lower)alkylsulfonyl,(lower) alkyl and (lower)alkoxy and nontoxic, pharmaceuticallyacceptable salts thereof.

The nontoxic, pharmaceutically acceptable salts include metallic saltssuch as sodium, potassium, calcium and aluminum, the ammonium salt andsubstituted ammonium salts, e.g., salts of such nontoxic amines astriallrylamines, including triethylamine, procaine, dibenzylamine,N-benzyl beta phenethylamine, l-ephenamine,N,N'-dibenzylethylenediamine, dehydrobietylamine, N,N'- bisdehydroabietylethylenediamine, N (lower)alkylpiperidines, e.g.,N-ethylpiperidine, and other amines which have been used to form saltswith benzylpenicillin. The term (low/er) alltyl as used herein meansboth straight and branched chain aliphatic hydrocarbon radicals havingfrom one to ten carbon atoms such as methyl, ethyl, propyl, isopropyl,butyl, isobutyl, t-butyl, amyl, hexyl, Z-ethylhexyl, heptyl, decyl, etc.Similarly, where the term (lower) is used as part of the description ofanother group, e.g., (lower) alkoxy, it refers to the alkyl portion ofsuch group which is therefore as described above in connection with(lower) alkyl. Also included within the scope of the present inventionare easily hydrolyzed esters and amides which are converted to the freeacid form by chemical or enzymatic hydrolysis.

The preferred compound of the present invention has the formula abovewherein R and R are each hydrogen and is named6-(2-phenylpyrazine-3-carboxarnido)peni cillanic acid.

The products of the present invention are prepared by the reaction of6-aminopenicillanic acid, preferably in the form of a neutral salt suchas the sodium salt or the triethylamine salt, With an acid chloridehaving the formula wherein R and R have the meaning set forth above, orits functional equivalent as an acylating agent for a primary aminogroup. Such equivalents include the corresponding carboxylic acidbromides, acid anhydrides and mixed anhydrides with other carboxylicacids, includ ing monoesters, and particularly lower aliphatic esters,of carbonic acid. In addition, an acid azide or an active ester orthioester (e.g., with p-nitrophenol, thiophenol, thioacetic acid) may beused or the free acid itself may be coupled with 6aminopenicillanic acidby the use of enzymes or of a carbodlimide reagent [cf. Sheehan andHess, J. Amer. Chem. Soc.. 77, 1067 (1955)]. The methods for carryingout these reactions to produce a penicillin and the methods used toisolate the penicillins so produced are well-known in the art.

The novel 2-phenylpyrazine-3-carboxylic acids used to produce thecompounds of the present invention are prepared by acid hydrolysis,e.g., heating in sulfuric acid, of the corresponding2-phenyl-3-cyanopyrazines. These acids are converted to their reactivederivatives, when desired, by the usual methods, e.g., to the acidchloride by heating with thionyl chloride.

The 2-phenyl-3-cyanopyrazines are prepared by the method used by Karmaset al. [1. Amer. Chem. Soc. 74, 1580-4584 (1952), and 78, 2141-2144(1956)], to prepare 2-phenyl-3-cyanopyrazine itself by refluxing2-phenyl-S-brornopyrazine (also called 2-bromo-3-phenylpyrazine) withabout three moles of cuprous cyanide in y-picoline, quenching thereaction mixture in cold, aqueous acid (e.g., 4 N, HCl), and extractingthe nitrile with an organic solvent such as chloroform from which it isthen distilled or crystallized.

The intermediate 2-bromo-3-phenylpyrazines are prepared as described inthe literature, e.g., by Karmas et al. (ibid) or by R. G. Jones [J.Amer. Chem. Soc., 71, 78-81 (1949), and references therein and US.Patent 2,520,088], as, for instance, by the following reaction scheme:

@omooon R2 NHa ino-002115 R1 Br Bra; 0012; 0211505 wherein R and R havethe meaning set forth above.

n -3 Alternatively, the Z-phenylpyrazine-3-carboxylic acids are preparedby the methods described in the examples below, e.g., by conversion ofthe appropriate acetophenone to an ester of the substitutedbenzoylacetic acid and thence to the ester of the substitutedbenzoylglyoxylic acid and then to the substitutedZ-phenylpyrazine-B-carboxylic acid.

The following examples will serve to illustrate this invention withoutlimiting it thereto.

A mixture of 272 g. (2 moles) of phenylacetic acid and 350 ml. (4.8moles) of thionyl chloride was refluxed for two hours. This reactionmixture was stirred and 340 g. (4.25 moles) of bromine was addeddropwise at about 85 C. over a period of one hour. After addition,stirring was continued for 4 hours under reflux. The reaction mixt-urewas cooled in an ice bath and 500 ml. of ethanol was added with vigorousstirring and the mixture was allowed to stand at room temperatureovernight. Cold water was added with vigorous agitation and the organiclayer was removed. The aqueous layer was extracted twice with ether. Theorganic layer and the ether extracts were combined, dried over calciumchloride and distilled to give 454 g. ethyl e-bromophenylacetate, 13.1.141-144 C./15 mm.

Ethyl u-bromophenylacetate (49 g., 0.2 mole) was dis solved in 100 ml.of ethanol, cooled to to 5 C. and ammonia gas was bubbled in for fivehours. A white precipitate separated at first and then graduallydissolved to give a yellow solution. After standing at 0 to 5 C. forfive days, the mixture was treated with active carbon and filtered. Thecarbon cake was washed with 100 ml. of ethanol. The washings werecombined with the filtrate and concentrated to about 50 ml. in vacuo toprecipitate the product as a white solid, which was filtered, washedwith 50 ml. of ethanol and dried in vacuo. The yield of the product,phenylglycineamide hydrobromide, was 23.5 g., M.P. 268-272 C. (dec.) (insealed tube).

A suspension of 116 g. (0.5 mole) of phenylglycineamide hydrobromide in600 ml. of methanol was chilled at -40 C., a solution of g. of sodiumhydroxide in 50 m1. of water was added and 90 g. of 40% glyoxal wasadded dropwise below C. A solution of 25 g. of sodium hydroxide in 50ml. of water was again added dropwise to the mixture and the reactionmixture was kept at 5 C. for two hours and then at room temperature forseveral hours. Addition of 50 ml. of 12 N HCl gave a white precipitateof the product, which was collected by filtration and extracted byboiling chloroform. The chloroform solution was evaporated to drynessand the residue was crystallized from ethyl acetate to give 60 g.Z-phenyl-S-hydroxypyrazine melting at 172-174 C.

A mixture of 55 g. (0.34 mole) of the 2-phenyl-3- hydroxypyrazine and120 ml. of phosphorous tribromide was stirred and heated at 180 C. forfour hours. The dark brown reaction mixture was poured onto crushed iceand extracted with chloroform. The extract was treated with activecarbon, dried over anhydrous sodium sulfate and evaporated to dryness.The residue was recrystallized from ethanol to give 44.5 g. ofZ-phenyl-S-bromQ- pyrazine, M.P. 88-895 C.

A mixture of 11.3 g. (0.05 mole) of the 2-phenyl-3- brornopyrazine and15 g. of cuprous cyanide in ml. of q-picoline was heated under refluxand then poured into a cold mixture of 400 m1. of 4 N hydrochloric acidand 100 ml. of chloroform and filtered. The filter cake was thoroughlywashed with chloroform. The filtrate and the washings were combined,treated with active carbon and dried over sodium sulfate.2-phenyl-3-cyanopyraz1ne was crystallized by evaporating the solvent.Yield 6 -g., M.P. 94-96 C.

Analysis.--CalCd. for C H N C, 72.91; H, 3.89; N, 23.19. Found: C,72.83, 72.45; H, 3.76, 3.47; N, 22.73, 22.72. v 2228 cmr A mixture of 6g. (0.033 mole) of the 2-phenyl-3-cyanopyrazine and 25 ml. of cone.sulfuric acid was heated at about 120 C. for three hours and chilled inan ice-salt bath. A solution of 4.2 g. of sodium nitrite in 10 ml. ofwater was added dropwise maintaining the temperature of the solutionbelow 20 C. After addition was completed, the mixture was heated on awater bath for three hours and poured onto crushed ice. A precipitatewas collected by filtration and Washed with water. This product was themonohydrate of 3-phenylpyrazine-2-carboxylic acid. Yield 6 g., M.P.74-76 C. u 1685 cm.

Analysis.-Calcd for C H N O H O: C, 60.54; H, 4.62; N, 12.84. Found: C,60.12, 60.33; H, 4.60, 4.40; N, 12.51, 12.76. Recrystallization frombenzene gave its anhydrous acid. M.P. 141-142 C., 11C:01735 cm.-

Analysis.-Calcd. for C11H8N202: C, 65.99; H, 4.38; N, 13.99. Found: C,65.87;H, 3.97;N, 13.78.

A mixture of 11 g. (0.05 mole) of the 3-phenyl-pyrazine-Z-carboxylicacid and 25 ml. of thionyl chloride was refluxed for one hour. Thereaction mixture was evap orated to remove excess of thionyl chloride.The dark oily residue wa distilled to give 9.5 gm. 3-phenylpyrazine-2carboxylic acid chloride, B.P. 142-145 C./4 V

A suspension of 10.8 g. (0.05 mole) of 6-APA (6- aminopenicillanic acid)in ml. of water and 50 ml. of acetone was cooled below 0 C. and stirred.A soltitiod of 2 g. (0.05 mole) of sodium hydroxide in 20 ml. of waterwas added to this suspension. The 6-APA dissolved and a pale yellowsolution was obtained. Then there was added 8.4 g. (0.1 mole) ofpowdered sodium bicarbonate. A solution of 10.9 g. (0.05 mole) of 3-phenylpyrazine-Z-carboxylic acid chloride in 20 ml. of dry acetone wasadded dropwise at 0-2 C. to the 6 aminopenicillanate solution over aperiod of 20 minutes. After addition was completed, the mixture wasstirred at 5-7 C. for one hour. There was added 300 ml. of water below10 C. The aqueous layer, which was separated and washed with ether andethyl acetate successively, was layered with 300 ml. of ethyl acetateand acidified to pH 2 by phosphoric acid (3:1) below 5 C. The aqueousphase was again extracted twice with 200 ml. portions of ethyl acetate.The ethyl acetate layer was combined with ethyl acetate extracts, washedwith ml. of cold water and dried over anhydrous sodium sulfate. To thefiltrate was added 15 ml. of 48% sodium 2-ethylhexanoate solution inmethyl isobutyl ketone. S0 dium6-(3'-phenylpyrazine-2'-carboxamido)penicillanate which separated wascollected by filtration, washed with ether and dried in vacuo. Yield 18g. (82% as monohydrate). This product was found to inhibit Staph.aurezls Smith at 0.39 mcg./ml., to inhibit Staph. aureus BX-1633, whichis highly resistant to benzylpenicillin, at 1.56 meg/ml, to exhibitversus Staph. aureus BX-l633 in mice a CD of 10 mgmJkg. uponintramuscular injection (compared to 2. CD50 of 25 mgm./kg. foroxacillin) and to be quite stable to acid (having a half-life of 90minutes at pH 2 and 37 C. compared to 7 minutes for benzylpenicillin ina comparative experiment).

EXAMPLE 2 In the procedure of Example 1 there is substituted for thephenylacetic acid an equimolar amount of the acid chloride prepared bytreatment with thionyl chloride of 4-trifluoromethylphenylacetic acid,Z-methylphenylacetic acid, 3-methoxyphenylacetic acid,3,4-dimethoxyphenylacetic acid, 2,6-dimethoxyphenylacetic acid,2,4-dimethylphenylacetic acid, 2,6-dimethylphenylacetic acid, and4-isopropylphenylacetic acid,

respectively to produce the acids,

respectively, which are isolated as their water-soluble sodium salts andfound to contain the 18-lactam structure as shown by infra-red analysisand to inhibit gram-positive bacteria, e.g., Staph. aureus, at lowconcentrations.

EXAMPLE 3 Method of preparation sodium 6- [3-(0-chl0r0phenyl)pyrazine-Z-carboxamido] penicillanate (A) 2-(o-chlorophenyl)pyrazine-3-carboxylic acid.

01 ([31 o112o1 @omon B.P. 138143 O./30 mm.

12.12. 130140 042-3 mm. M.P. 96 0.

U oHoNHi I OH h/LP. 171-l73 C.

M.P. 135 C./0.5 mm. B.P. LEA-137 O./0.60.7 mm.

("J-NH: l looon-nzo M.P. 172 C. M.P. 74-78 C.

c-Chlorophenylacetic acid was prepared from o-chlorobenzylchloride asillustrated above. u-Bromination and esterification ofo-chlorophenylacetic acid were carried out in the usual manner.Treatment of the a-bromoester with cone. ammonium hydroxide or bybubbling of gaseous ammonia into an alcoholic solution of the abromoester gave the corresponding glycineamide hydrobromide. A sample ofglycine amide hydrobromide was treated with glyoxal at -15 C. and2-(o-chlorophenyl)-3-hydroxypyrazine was obtained.

The yield of 2-o-chlorophenyl-3-hydroxypyrazine was improved byperforming the reaction of glycinamide with 'glyoxal at 40 C. using DryIce-acetone mixture.

The 2-(o-chlorophenyl)-3-hydroxypyrazine was converted to2-(o-chlorophenyl)-3-'bromopyrazine by refiuxing with a large excess ofphosphorous tribromide. Conversion of 2-(o-chlorophenyl)-3-bromopyrazineinto the corresponding nitrile was carried out by using an equivalentamount of cuprous cyanide. Fractionation of the reaction mixture gave2-(o-chlorophenyl-3-cyanopyrazine, boiling at 130135 C./0.S mm.

Analysis.-Calcd for C H ClN C, 61.26; H, 2.80; N, 19.51. Found: C,59.70, 59.54; H, 2.78, 3.01; N, 16.24, 16.20.

These values seemed to indicate that the liquid contained a small amountof brornopyrazine. Hydrolysis of this cyanopyrazine with hydrochloricacid was not successful. When the cyanopyrazine was treated withhydrogen peroxide in sodium hydroxide solution,2-(o-chlorophenyl)pyrazine-3-carbonyl amide (MP. 172 C.) was obtained ina good yield. Reaction of the cyanopyrazine with sodium carbonate alsoafforded larger amounts of the amide as well as a trace of2-(o-ch1orophenyl) pyrazine-3-carboxylic acid. The acid was alsoobtained by treating 2-(o-chlorophenyl)pyrazine-3-carbonyl amide withnitrous acid. Conversion of the pyrazinamide into2-och1orophenylpyrazine-3-carboxylic acid was alternatively carried outby the action of nitrous acid in concentrated hydrochloric acid and bythe use of aqueous potassium hydroxide. The latter *gave a quantitativeyield. Monohydrate, M.P. 74-78 C. Anhydrous acid, M.P. 138-140 C. Calcdfor C H ClN O C, 56.30; H, 3.01; N, 11.94. Found: C, 56.31, 56.50; H,3.06, 2.94; N, 12.02, 12.12.

(B) Sodium 6-[3-(o-chlorophenyl)pyrazine-Z-carboxamido]penicillanate.

2-(o-chlorophenyl)pyrazine carboxylic acid was converted to the acidchloride with thionyl chloride. A small amount of the acid chloride wasesterified to the corresponding ester.

Analysis.-Calcd for C H ClN O C, 59.43; H, 4.22; N, 10.67. Found: C,59.34; H, 4.09; N, 10.94, 10.89.

The remain of the acid chloride was used for condensation with6-aminopenicillanic acid (6APA). Assay of the penicillanate: NH OH, 924meg/mg; iod. 835 meg/mg; cup, 122 ../mg.

Analysis.Calcd for C19H15ClN404SNa'H20: C, 48.25; H, 3.84; N, 11.84.Found: C, 48.79, 49.49; H, 3.95, 3.97; N, 11.18, 11.29.

This product was found to inhibit Staph. aureus Smith at about 0.4mcg./ml., to inhibit Staph. aureus BX-1633, which is highly resistant tobenzylpenicillin at about 3-12 meg/ml, to exhibit versus Staph. aureusBX-1633 in mice a CD of about 45-82 mgm./kg. upon intramuscularinjection and to be quite stable in acid (having a half-life of 11.3hours at pH 2 and 37 C. compared to 10 minutes for benzylpenicillin in acomparative experiment).

EXAMPLE 4 Method of preparation of sodium 6-[3-(p-chl0r0phenyl)pymzine-Z-carboxamido] penicillanate 18.1. l29136 C./2.5 mm.

Ell-O C2115 C O OH not isolated Ml. 74 76 C.

M.P. 83-87 C.

S CH3 t I \N/ (fi-I\H()HCH 0-011;

0 O=CN CHCOONa p-Chloroacetophenone was prepared by Friedel-Craftreaction of chlorobenzene and it was then reacted with ethyl carbonatein the presence of sodium ethoxide to give ethyl p-chlorobenzoylacetatewhich was oxidized with selenium dioxide to give ethylp-chlorobenzoylglyoxalate. An equimolar mixture ofp-ohlorobenzoylglyoxalate, ethylenediamine monohydrochloride 'and sodiumacetate in alcohol was heated under reflux for 23 hours. After removingthe solvent by evaporation under reduced pressure, the residue was takenup in ether and the ethereal solution was washed with aqueous sodiumbicarbonate. Evaporation of the solvent afforded crude ethyl2-p-chlorophenylpyrazine-3-carboxylate. Hydrolysis of the crude esterwith alcoholic potassium hydroxide gaveZ-p-chloropheny1pyrazine-3-carboxylic acid hydrate. A suspension of2-p-chlorophenylpyrazine-3-carboxylic acid monohydrate and thionylchloride in dry methylene chloride was refluxed for two hours on a waterbath. After the solvent and excess thionyl chloride were removed bydistillation under diminished pressure, the crude acid chloride wasdissolved in dry benzene and precipitated by adding petroleum ether. Theacid chloride (3.7 g., 0.015 mole) was reacted with 6-APA inacetone-water in the usual manner to produce sodium6-(2-p-chlorophenylpyrazine-3-carboxamide)penicillanate.

This product was found to inhibit Staph. aureus Smith at about 1.0-1.6mcg./ml., to inhibit Staph. aureus BX-l633, which is highly resistant tobenzylpenicillin, at about 3.16.3 meg/ml. and to exhibit versus Staph.aureus BX-1633 in mice a CD of about mgm./kg. upon intramuscularinjection.

EXAMPLE 5 M ethod of preparation of sodium 6-[3- (p-metho'xyphenyl)pyrazirze-Z-carboxam ido] penicillanate omo CHsO-COCH3 it ucam-@o-orn-o-00m,

i it i omo-Q-co- 0-0 02115 j: OOH;

via acid chloride OGHa I /S\ /OH: C;C H3

vacuo. Oxidation of the wmethylene group of the ester was carried outusing selenium dioxide (13.1 150-157 C./ 2 mm.).

2-(p-rnethoXyphenyDpyrazine 3-carboXylic acid Was prepared by thereaction of p-methoxybenzoylglyoxylate and ethylenediaminedihydrochloride in alcohol in the presence of sodium acetate. EthylZ-(p-methoxyphenyD- 5,6-dihydropyrazine-3-carboxylate was not isolated,but the desired pyrazinecarboxylic acid and p-anisic acid were obtainedby hydrolysis of the oily product with alcoholic potassium hydroxide.

Analysis.Calcd for C H N O -H O: C, 58.06; H, 4.87; N, 11.27. Found: C,58.07; H, 4.80; N, 11.20, 11.07.

Dehydrogenation seemed to occur in the course of reaction ofa,[3-diketoester with diamine. The acid was converted to the acidchloride with thionyl chloride in dry benzene. The excess of thionylchloride and the solvent were distilled ofi in vacuum.

The acid was dried by azeotropic distillation with benzene. Thionylchloride Was also used in methylene chlo ride to prepare the acidchloride of this acid; the resulting acid chloride Which showed thecarbonyl absorption at 1765 (3111." in' IR spectrum was reacted with6-APA by the usual acid chloride method to yield sodium 6-[3-(p- 133methoxyphenyDpyrazine-Z carboxamido] penicillanate as an almostcolorless powder. The carboxylate band (1605 cm. of this penicillin wasquite strong as compared with the fi-lactam band (1770 cm. and the sidechain amide band (1670 cmr It seemed to be due to overlap with phenylskeletal vibration which was enhanced by the methoXyl group attached tothe ring.

A suspension of 2-(p methoxyphenyl)pyrazine-S-carboxylic acid hydrate inmethylene chloride was refluxed with thionyl chloride for four hours andZ-(p-methoxyphenyl)pyrazine-3-carbonyl chloride was obtained as a yellowprecipitate melting at 8388 C., yield 4.8 g. (85%). The acid chloridewas combined with 6-APA in an aqueous acetone solution to give thesodium salt of the penicillin, yield 3.0 g. Assay: NH OH, 709 mcg./mg.;iod., 710 rncg./mg.; cup, 65,u./mg.

This product was found to inhibit Staph. aureus Smith at about 0.8mcg./ml., to inhibit Staph. aureus Bil-1633, which is highly resistantto benzylpenicillin, at about 1.5 6- 3.1 meg/ml, to exhibit versusStaph. aureus BX-1633 in mice a CD of about 35-45 mgm/kg. uponintramuscular injection and to be quite stable in acid (having ahalflife of 7.75 hours at pH 2 and 37 C. compared to 10 minutes forbenzylpenicillin in a comparative experiment).

EXAMPLE 6 Method of preparation of sodium6-[3-(p-methylzkiophenyl)pyrazine-2-carboxamido]pelzicillmzate and s0-dium 6- [3- (p-methylszilfonylphenyl)pyrazine-Z-carboxamz'd01penicillanate IB.P. ss-w 0 15 mix M.P. 81 C.J'MQP. 165-175 0. (162.)

H ll

B.P. -155 C./0.50.6 mm.

Thiophenol was prepared from benzenesulfonyl chloride according to themethod described in Organic Synthesis Col. vol. I, p. 504. Thioauisolwas acetylated with acetic anhydride in the presence of anhydrousaluminum chloride according to Noller and Adams [3. Amer. Chem. Soc.,46, 1892 (1924)] to give p-methylthioacetophenone which was reacted withdiethyl oxalate and sodium ethoxide in the usual manner employed in thepreparation of p-methoxy and p-chlorobenzoylacetates.

Ethyl p-methy1thiobenzoylacetate was successfully isolated asgreen-colored copper salt. The copper salt was decomposed with aceticacid to give free ethyl p-methylthiobenzoylacetate, which was oxidizedwith selenium dioxide in dioxane. The IR spectrum of the residual oilafter removing selenium and solvent, shows formation ofp-methylthiobenzoylglyoxylate. Broadening of carbonyl bands both at 1745and 1675 C111.' 1 and appearance of a new band at 920 cm." as comparedwith the spectrum of ethyl p-methy1thiobenzoylacetate are the sametendency as in the case of conversion from ethyl p-methoxybenzoylacetateto ethyl p-methoxybenzoylglyoxylate.

A solution of ethyl p-methylthiobenzoylglyoxylate (2.5 g., 0.01 mole) in40 ml. of ethanol was reacted with a solution of an equimolarethylenediamine in 20 ml. of ethanol at 2 C. for three hours withstirring and 2.5 g. of a white precipitate melting at 8889 C. wasobtained. The IR spectrum has an ester carbonyl band at 1730 cm.- and acomplicated band at about 3300 cm.- due 12 to NH and/ or OH, and acarbonyl band of benzoylglyoxylate at 1740 cm." and 1670 cm?disappeared. This fact suggests formation of an intermediatehydropyrazine ring.

Condensation of p-methylthiobenzoylglyoxalate with ethylenediamine gaveintermediate compound A or B, both of which were converted into compoundC by recrystallization from ethanol. Each of compounds A, B and C wasconverted to 2-(p-methylthiophenyl)pyrazine- S-carboxylic acid throughintermediate compounds D and E as shown in Chart I. Experimentalconditions are given in Table II and the properties of compounds A-E inTable 1. Compound E was identified as ethyl2-(p-methylthiophenyl)-pyrazine-3-carboxylate, which was also preparedfrom the corresponding acid chloride and ethanol. The pyrazine acid wasobtained as a monohydrate, which was converted by recrystallization frombenzene into a product solvating one mole of benzene. M.P. 75-80 C.

Analysis.Calcd for C H N O S-C H 1 C, H, 4.97; N, 8.64. Found: C, 66.30,66.64; H, 5.05, 5.09;

N, 8.65, 8.86. Hydrate:

are 305 mu (E 16,200), v 1680 emf- Benzene solvate:

6 14,200 v 1725 GEL-1.

TABLE I 11.1. C.) Compd. Compd. Compd. Compd. Compd.

A, 87-90 B, 94 C, 108-111 D, oil E. oil

3, 310 r 0 (cm.- g, 3, 300 3, 2'80 None None v0.0 (cmr 1, 730 1, 755 1,740 1, 735 UV 1,700

E23 111,. 314 No data 270. 5 gig 306 E 1% 210 535 1 cm. 494 333 i 185Eugene TABLE II First Step Second Step Third Step Expt. Starting N 0.Material Reagents and Reagents and Reagents and Yield of ConditionsProduct Conditions Product Conditions Pyrazine Acid 213-101-- Compd. B,EtOH 50 1111., reflux, Compd. D EtGH ml., KOH 0.5 Pyrazine 100 mg.

0.5 g. overnight. g., reflux, 4 hrs. Acid. 213-108--- Compd. B, EtOH 200ml., reflux, Compd. D EtOH 160 ml., KOH Pyrazine 130 mg.

2 g. 10 hrs. 1 g., reflux, 4 hrs. Acid. 13 9 Compd, A, Benzene 120 ml.,reflux, Compd. D-... 0. H01 100 ml., H20 200 1 g. overnight. ml., roomtemp. 2131l0 Compd. A, Benzene 100 ml., reflux, Compd. D- EtOH ml., KOH0.5 I

1 g. 3.5 hrs. g., reflux, 3 hrs. 213111 Compd. A, EtOH ml., reflux,Compd. D EtOH 50 ml., KOH 0.5

0.5 g. 15 hrs. g., reflux, 3.5 hrs. 213l12 Compd. A, Benzene 50 ml.,Compd. D Benzene 50 ml., Compd. E EtOH 50 ml., KOH 0.5 367 mg.

0.5 g. Pyridine 1 drop, re- Alumina 5 g., room g., reflux, 3 hrs.

flux, 15 hrs. 2 hrs. 213113 Compd. A, Benzene 50 ml., Compd. D Benzene50 ml., Compd. E EtOH 50 ml., KOH 0.5 283 mg.

1 g. Alumina 5 g., room Aluminafi g., room g., reflux, 3 hrs.

temp., 2 hrs. temp., 2 hrs. 213-115"- Compd. C, EtOH 50 ml., KOH 0.5

0.5 g. g., reflux, 4 hrs. 213116 Compd. C, Benzene 100 ml., reflux,Compd. D Benzene 100 ml., Compd. E EtOH 100 ml., KOH 400 mg. 2 g. 4 hrs.w Alumina 10 g., room 1 g., reflux, 3.5 hrs.

temp., 2.5 hrs. 213117 Compd. A, Benzene 100 ml., reflux, Compd. DBenzene 100 ml., Compd. 13.... EtOH 100 ml., KOH 557 mg. 2 g. 3 hrs.Alumina 10 g. room 1 g., reflux, 3 hrs.

temp., 2 hrs. 213-118-.- Compd. A, Benzene 300 ml., reflux, Compd. DBenzene 300 ml., Compd. 11.... EtOH 300 ml., KOH 2.18 g. 6 g. overnight.Alumina 30 g., room 3 g., reflux, 3 hrs.

temp., 3.5 hrs. 213-132.-- Compd C, EtOH 20 ml., SeOz 0.4 Compd. E 5

1.8 g. g., room temp., 3 hrs.

p-Methylthiobenzoylglyoxylic acid was recovered.

, CHgNH CH S COCOCOOH I CEQNHZ in ethanol at C.

Compound A Compound B reflux in reflux in ethanol ethanol Compound 0reflux in reflux in benzene 2 53 benzene or EtOH or EtOH stirred in EtOHwith Compound D 59.02 at room temperature stirred in benzene withalumina at room temperature Compound E N 3 C OOEt ir reflux in R E t OHalc Kori SCH: S0012 I Q SCH:

C O OH C 001 N 1% Chart I 2 (p methylthiophenyl)pyrazine 3 carboxylicacid (benzene solvate) was converted into the corresponding acidchloride, which was reacted with 6-APA in the usual manner inacetone-water to give sodium 6-[3-(p-methylthiophenyDpyrazine 2carboxamido1penicillanate. Assay: NH OH, 762 meg/mg; iodine, 526mcg./mg.; cup, 54 .t./ mg. This product was found to inhibit Staph.aureus Smith at about 0.78-0.156 meg/ml, to inhibit Staph. aureusBX-i633, which is highly resistant to ben- Zylpenicillin, at about1.56-3.13 mcg./ml. and to exhibit versus Staph. aureus BX-1633 in mice aCD of about 13 mgm./ kg. upon intramuscular injection.

2 (p methylthiophenyl) pyrazine 3 carboxylic acid (monohydrate) wasrepeatedly prepared and then oxidized with potassium permanganate toZ-(p-methylsulfonylphenyl)pyrazine-3-carboxylic acid, which was obtainedin various forms according to conditions of precipitation andrecrystallization solvents as shown in the following table.

The 2 (p methylsulfonylphenyl)pyrazine 3 carboxylic acid was convertedvia its acid chloride in the usual manner to sodium6-[3-(p-methylsulfonylphenyl)- pyrazine-Z-carboxamido]penicillanate.This product was found to inhibit Staph. aureus Smith at about 1.56mcg./ ml., to inhibit Staph. aureus BX-1633, which is highly resistantto benzylpenicillin, at about 3.13 mcg./ml., to exhibit versus Staph.aureus Smith in mice a CD of about 6.25 mgm./kg. upon intramuscularinjection and to be quite stable in acid (having a half-life of greaterthan five hours at pH 2 and 37 C. compared to 15 minutes forbenzylpenicillin in a comparative experiment).

While in the foregoing specification various embodiments of thisinvention have been set forth in specific detail and elaborated for thepurpose of illustration, it will be apparent to those skilled in the artthat this invention is susceptible to other embodiments and that many ofthe details can be varied widely without departing from the basicconcept and the spirit and scope of the invention.

We claim:

1. A compound selected from the group consisting of an acid of theformula N\ I I 0254mm...

N CH3 wherein R and R represent (lower) alkyl.

3. A compound of the formula wherein R and R represent (lower) alkoxy.

A-Product precipitated by acidification of a diluted solution orrecrystallized from water.

BProduct recrystallized from ethanol.

C-The product precipitated by acidification of a concentrated solutionmelted at 166- 167 C.

l5 4. Acompound of the formula wherein R and R represent chloro. 5. Acompound of the formula 0 s CH:

wherein R represents (lower) alkyl.

6. A compound of the formula 0 S /N\ NH-CHC wherein R represents chloro.

7. A compound of the formula wherein R represents (lower) alkoxy.

15 8. A compound of the formula 0 s CHa /N\ "3-Nrr-oH-c c L l l I 1O=CNOHCOOH References Cited by the Applicant UNITED STATES PATENTS2,520,088 8/1950 Jones. 2,941,995 6/1960 Doyle et a]. 2,951,839 9/1960Doyle et a1.

OTHER REFERENCES Jones: J. Amer. Chem. Soc. 71, 78-81 (1949).

Karmas and Spoerri: J. Amer. Chem. Soc. 74, 1580- 1384 (1952).

Karmas and Spoerri: J. Amer. Chem. Soc. 78, 2141- NICHOLAS S. RIZZO,Primary Examiner.

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF AN ACID OF THEFORMULA